Fucose-containing sulfated polysaccharides from brown seaweeds inhibit proliferation of melanoma cells and induce apoptosis by activation of caspase-3 in vitro

Abstract

Fucose-containing sulfated polysaccharides (FCSPs) extracted from seaweeds, especially brown macro-algae, are known to possess essential bioactive properties, notably growth inhibitory effects on tumor cells. In this work, we conducted a series of in vitro studies to examine the influence of FCSPs products from Sargassumhenslowianum C. Agardh (FSAR) and Fucus vesiculosus (FVES), respectively, on proliferation of melanoma B16 cells and to investigate the underlying apoptosis promoting mechanisms. Cell viability analysis showed that both FCSPs products, i.e., FSAR and FVES, decreased the proliferation of the melanoma cells in a dose-response fashion, with FSAR being more potent at lower dosages, and FVES being relatively more anti-proliferative than FSAR at higher dosages. Flow cytometric analysis by Annexin V staining of the melanoma cells exposed to the FCSPs products confirmed that both FSAR and FVES induced apoptosis. The FCSPs-induced apoptosis was evidenced by loss of plasma membrane asymmetry and translocation of the cell membrane phospholipids and was accompanied by the activation of caspase-3. The FCSPs bioactivity is proposed to be attributable to distinct structural features of the FCSPs, particularly the presence of sulfated galactofucans (notably in S.henslowianum) and sulfated fucans (notably in F. vesiculosus). This study thus indicates that unfractionated FCSPs may exert bioactive effects on skin cancer cells via induction of apoptosis through cascades of reactions that involve activation of caspase-3.

Keywords: anti-tumor; apoptosis; bio-activity; fucoidan; fucose; sulfated polysaccharides.

Figure 1

Infrared analysis of fucose-containing sulfated polysaccharides (FCSPs) from (a) Sargassum henslowianum C. Agardh (FSAR) and (b) Fucus vesiculosus (FVES) scanned between 1800 and 550 cm⁻¹.

Figure 2

One-dimensional ¹H NMR spectra of crude FCSPs from (a) Sargassum henslowianum C. Agardh (FSAR) and (b) Fucus vesiculosus (FVES) in D₂O obtained using an INOVA 600 NMR spectrometer (Agilent Technologies, Tokyo, Japan).

Figure 3

MTT based colorimetric assay of cell viability of melonama B16 cells after treatment for 24 h with different dosage levels of crude fucose-containing sulfated polysaccharides from Sargassum henslowianum C. Agardh (FSAR) and Fucus vesiculosus (FVES), respectively. Cell density was 6 × 10⁴ cells per well. ^a,b indicate statistically significantly different (P ≤ 0.05) cell viability levels after treatment with the two FCSP products at the same dosage level (mg/mL) (n = 4).

Figure 4

Flow cytometric analysis by Annexin V staining of Melanoma B16 cells treated for 24 h with 0.2 mg/mL crude fucose-containing sulfated polysaccharide (FCSP) products extracted from S. henslowianum C. Agardh (FSAR) and F. vesiculosus (FVES), respectively. (a) Apoptosis induced by FSAR (41 ± 3%, n = 2), FVES (30 ± 5%, n = 2); control 11.66 % (data not shown) (b) FSAR data and (c) FVES data for FACS scans of FCSP treated Melanoma 16 cells that were viable and not undergoing apoptosis (Annexin V⁻ and 7-AAD⁻); undergoing early apoptosis, with membrane integrity intact (Annexin V⁺and 7-AAD⁻); in the latest stage apoptosis and dead (Annexin V⁺ and 7-AAD⁺ ), respectively.

Figure 5

Activation of caspase-3 after treatment of melanoma B16 cells with different dosages of FCSPs from S. henslowianum (FSAR) and F. vesiculosus (FVES). For each dosage treatment the caspase-3 activity was assayed on a cytosolic extract of melanoma B16 cells with a DEVD-pNA substrate (contact at 37 °C for 1 h) and spectrophotometric detection by measuring the absorbance at 405 nm (n = 2).

Figure 6

Proposed mechanism for inhibition of the proliferation of melanoma cells by FCSPs: Activation of macrophages via membrane receptors, which leads to the production of cytokines that enhance NK cell activation. Activated NK cells release Granzyme B and perforin through granule exocytosis into the space between NK cells and melanoma cells to initiate caspase cascades in melanoma cells. Assimilation of Granzyme B by the tumor cells is facilitated by perforin. Granzyme B then initiates apoptosis by triggering the release of mitochondrial cytochrome c and apoptosome formation leading to caspase-3 activation, which in turn translocates the nucleus causing DNA fragmentation—the distinct morphological change of cells by apoptosis [36,37].